International Journal of Molecular Sciences Review Microtubule Dysfunction: A Common Feature of Neurodegenerative Diseases Antonella Sferra *, Francesco Nicita and Enrico Bertini Unit of Neuromuscular and Neurodegenerative Disorders, Genetics and Rare Diseases Research Division, Bambino Gesù Children’s Hospital, IRCCS, 00146 Rome, Italy; [email protected] (F.N.); [email protected] (E.B.) * Correspondence: [email protected] or [email protected]; Tel.: +39-06-6859-2104 Received: 8 September 2020; Accepted: 1 October 2020; Published: 5 October 2020 Abstract: Neurons are particularly susceptible to microtubule (MT) defects and deregulation of the MT cytoskeleton is considered to be a common insult during the pathogenesis of neurodegenerative disorders. Evidence that dysfunctions in the MT system have a direct role in neurodegeneration comes from findings that several forms of neurodegenerative diseases are associated with changes in genes encoding tubulins, the structural units of MTs, MT-associated proteins (MAPs), or additional factors such as MT modifying enzymes which modulating tubulin post-translational modifications (PTMs) regulate MT functions and dynamics. Efforts to use MT-targeting therapeutic agents for the treatment of neurodegenerative diseases are underway. Many of these agents have provided several benefits when tested on both in vitro and in vivo neurodegenerative model systems. Currently, the most frequently addressed therapeutic interventions include drugs that modulate MT stability or that target tubulin PTMs, such as tubulin acetylation. The purpose of this review is to provide an update on the relevance of MT dysfunctions to the process of neurodegeneration and briefly discuss advances in the use of MT-targeting drugs for the treatment of neurodegenerative disorders. Keywords: microtubules; neurodegeneration; microtubule dysfunctions; microtubule-targeting compounds 1. Introduction Microtubules (MTs) are structurally and functionally important components of the cell cytoskeleton, providing most of the spatial organization in the cell, and regulating different cellular functions such as intracellular trafficking, cell division, and organelle positioning. They are characterized by a great chemical and functional heterogeneity, and are thus subjected to highly dynamic regulatory mechanisms that modulate MT composition, tubulin chemical modification, and MT association with other proteins. The result of these regulations is the generation of MT arrays with different morphology and degrees of stability that are able to adapt to specific functions in accordance with the cell context. This modulation is particularly important in neurons, whose structures and functions strongly depend on the correct functioning of MTs. It is therefore not surprising that MT dysfunctions can be the cause or can contribute to the onset of neurodegenerative processes. Specific mutations in tubulin genes, dysfunctions of MT-associated proteins (MAPs), and alterations in the levels of tubulin PTMs are indeed associated with different forms of neurodegenerative diseases. This discovery has recently allowed the development of compounds aimed to counteract neurodegeneration by restoring the correct functioning of MTs. Although, recently, MT-oriented approaches currently represent one of the most promising strategies in the field of neurodegenerative diseases as several works have demonstrated that these compounds are effectively able to prevent or partially rescue MT dysfunctions in different animal and cellular models and that some of them have been translated into clinical trials. Int. J. Mol. Sci. 2020, 21, 7354; doi:10.3390/ijms21197354 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 7354 2 of 37 These results lead us to believe that in the near future, more preclinical approaches will be translated to patients, improving survival and their quality of life. 2. Microtubule Structural and Functional Complexity MTs are composed of heterodimers of α–β-tubulin that align head to tail to form linear protofilaments which associate laterally to form a hollow, polar cylinder [1]. The directional alignment of polarized α–β heterodimers into MTs make the structures intrinsically polarized because of the presence two distinct ends: a minus end that exhibits an α-tubulin subunit and the opposite plus end that exposes a β-tubulin subunit (Figure1). Figure 1. Microtubule organization in neurons. Microtubule (MT) organization is tightly regulated in the different neuronal compartments. In axon, MTs form stable, polarized bundles with uniform polarity orientation, exposing their plus minus ends away from the cell body. In proximal dendrites, MTs are organized in antiparallel bundles oriented with their plus ends pointing away or toward the soma. In the growth cone, MTs adopt four characteristic distributions: splayed, captured at the cortical matrix, looped, and bundled. At the top, MT structure (slide view and end view) is shown. In a solution of purified tubulin, MT elongation can occur to both ends [2]. In cells, MT growth is significantly more rapid at the plus end and occurs through the addition of α–β tubulin heterodimers in which each subunit of β-tubulin binds a GTP molecule. After heterodimer addition, the GTP is hydrolyzed to GDP and prolonged exposure of a terminal GDP at the α–β heterodimer of the plus end leads to MT depolymerization [3]. Within the cell, MTs do not reach a steady state length but undergo continual assembly and disassembly; the co-existence of growing and shrinking MTs in the same conditions is known as “dynamic instability”. Conversion from growth to shrinkage is defined “catastrophe”, whereas the switch from shrinkage to growth is called “rescue”. This “non equilibrium” in MT population is critical for several cell functions, such as cell division or during or the spatial arrangements of the cell [4]. MTs also undergo “treadmilling”, a dynamic process in which the plus end of the filament grows in length while the other one shrinks due to the removal of tubulin molecules bound to GTP from the minus end that travel to the plus end of the same MT [5]. In most cell types, MTs are nucleated at the centrosomal region in the MT-organizing center (MTOC), where a previously formed γ-tubulin ring complex provides a base for the start of filament Int. J. Mol. Sci. 2020, 21, 7354 3 of 37 extension [6]. The γ-tubulin ring complex has been proposed to form a ring-like template that binds the α-tubulin subunit exposed at the MT minus end. Thus, MTs grow with a defined polarity, with their minus ends anchored on the ring to γ-tubulin subunits and the plus end of the MT extending into the cytoplasm [7]. MTs constitute a heterogeneous and dynamic filament network with great chemical and functional complexity. This complexity is produced by three regulatory mechanisms: (i) differential incorporation of alternative isotypes of α and β tubulin into MTs; (ii) PTMs of these isotypes; (iii) MT interaction with MAPs. These mechanisms do not act separately but can influence each other. Each of these will be briefly discussed below and in the specific context of neuronal cells. In many organisms, both α and β tubulins are encoded by multiple genes at different loci [8,9]. Most of them, such TUBB2A, TUBB2B, and TUBA1A, TUBA1B, TUBA1C, are clustered in the genome and are presumably the products of genomic duplications [10]. In vitro experiments have shown that tubulin isotypes have different effects on MT dynamics [11–14], suggesting that they can differentially regulate the MT network in vivo. It was assumed that tubulin isotypes can assemble into discrete MT species that carry out unique functions [15]. This concept, known as the “multi tubulin hypothesis”, has long been quite controversial. It was demonstrated that all tubulin isotypes freely co-polymerize into heterogeneous MTs and that different α- and β-tubulin isotypes in fungi were functionally interchangeable [16,17]. However, several findings have shown that highly specialized MTs, such as ciliary axonemes [18,19], neuronal MTs [20,21], and MTs of the marginal band of platelets [22,23] depend on specific tubulin isotypes. Axonemal MTs of Drosophila olfactory neurons are highly enriched in β1- and β4-tubulins [24]. β6-Tubulin, instead, is essential for the formation of the platelet marginal band and the loss of this isoform results in thrombocytopenia [23]. Studies in Drosophila melanogaster have shown that specific tubulin isotypes are not functionally equivalent and possess distinctive properties depending on cell type and specific stages of development [25,26]. Hoyle and colleagues have demonstrated that in Drosophila male testes, the function of β2-tubulin is not rescued by the expression of β3-tubulin, and that the latter acts in a dominant-negative manner when co-expressed at levels exceeding 20% of the total tubulin. This finding indicates that while β2-tubulin regulates generic functions in Drosophila male testes, β3-tubulin has been evolved to ensure different MT / functions in fly [24]. Similarly, in mouse tubb3− − neurons the replacement of β3-tubulin by other β-tubulin isoforms cannot overcome its requirement in peripheral nerve regeneration, suggesting that the functions of the different β-tubulins are not interchangeable in neurons [27]. Tubulin isotypes also seem to directly determine MT structure by defining its protofilament numbers. In vitro experiments have shown that dimers of α1B-tubulin and β3-tubulin form typical MTs composed by 13 protofilaments, while heterodimers